Method for solubilization and naturation of somatotropins

ABSTRACT

A method for the solubilization and/or naturation of a somatotropin involves contacting a somatotropin with a detergent composition and water under conditions effective to obtain a naturated somatotropin, wherein the detergent composition may be a C 10 , C 12 , C 16  or C 18  acyl glutamate, a C 10 , C 14  or C 18  alkyl sulfate, an alcohol ethoxy sulfate, lauroyl ethylenediamine-triacetic acid (LEDA), a C 10  to C 18  linear alkyl benzene sulfonate, diphenyl disulfonate or an acyl amino acid.

This is a divisional of application Ser. No. 09/002,565, filed Dec. 31,1997 now U.S. Pat. No 6,034,224.

This application claims the benefit of U.S. provisional application Ser.No. 60/034,808, filed Dec. 31, 1996 now abandoned.

BACKGROUND OF THE INVENTION

Somatotropins are growth hormones which were originally discovered inpituitary gland extracts of various animals. Recombinant DNA technologyhas permitted the expression of somatotropins as heterologous proteinsfrom various host cells. Such recombinant somatotropins, e.g.,somatotropins produced in a microorganism such as E. coli bacteria thathas been transformed using recombinant DNA, are typically produced bythe host cell in a precipitated, denatured state having reduced orsubstantially no bioactivity. The absence of bioactivity is generallyattributed to the conformation of the recombinant somatotropin molecule,which lacks the formation of disulfide bonds. Recombinant somatotropinsare believed to be produced by the host cell in substantially reducedform (without disulfide linkages) due to the relatively high redoxpotential of host cells such as the E. coli cell. Most recombinantsomatotropins, such as bovine (bST) and porcine somatotropin (pST), arepackaged in the host cell as inclusion bodies, also referred to asrefractile bodies, which are cytoplasmic aggregates containing therecombinant somatotropin and oligomers thereof.

In order to recover the recombinant somatotropin in a bioactive state,the somatotropin, e.g., in the form of inclusion bodies, is preferablybut not necessarily isolated from the host cell, after which thesomatotropin may be solubilized to form somatotropin monomers, which maythen be naturated into a bioactive conformation. The naturatedsomatotropin may then be further purified to remove impurities such asother somatotropin species, e.g., oligomers such as dimers, and hostcell proteins, for example, by ion exchange to precipitate theimpurities (e.g., as described in U.S. Pat. No. 5,182,369, which isincorporated herein by reference) or other suitable techniques.

For example, Bentle et al., U.S. Pat. No. 4,652,630, which isincorporated herein by reference, refers to a method for solubilizationand naturation of somatotropin protein from inclusion bodies using anaqueous solution of urea to solubilize the inclusion bodies containingthe recombinant somatotropin. Bentle et al. reports using urea solutionshaving concentrations ranging from 2.5 to 7.5 M and a pH between about 9and 12 for the solubilization step. Once solubilized, the somatotropinprotein can be naturated according to the Bentle et al. method at analkaline pH.

Other methods have used a detergent to solubilize and naturate asomatotropin. For example, European Patent Specification publicationnos. 229,110 and 263,902 (The Upjohn Co.), which are incorporated hereinby reference, disclose a method for converting an insoluble form ofsomatotropin from a transformed microorganism to the native disulphidebond conformation by solubilizing and oxidizing the somatotropin in thepresence of a detergent. That method uses a detergent of sodiumdodecylsulfate (SDS) or a detergent of the formula:

CH₃—(CH₂)_(n)—CO—NR₁—CHR₂—COOH,

wherein n is 8 through 20 inclusive; R₁ is methyl or ethyl; and R₂ ishydrogen, ethyl, methyl, n-propyl or isopropyl. These specificationsfurther disclose a particular method for solubilizing recombinant bSTusing an aqueous solution of N-lauroyl methyl glycine, which isrepresented by the formula:

CH₃—(CH₂)₁₀—CO—NR₁—CHR₂—COOH,

in a sodium borate buffer at 0.1 to 0.5 M and a pH of 8 to 10.5. Aftersolubilization and naturation, European Patent Specification publicationnos. 229,110 and 263,902 state that the detergent is removed by an anionexchange resin.

Other publications also disclose methods for the solubilization andnaturation of recombinant somatotropins using various detergent andnon-detergent compounds including U.S. Pat. Nos. 4,677,196 and4,766,224, which also use SDS; U.S. Pat. No. 5,023,323, which uses SDSin combination with a chaotropic agent such as urea or guanidinehydrochloride; U.S. Pat. No. 5,240,834, which uses sarkosyl (N-lauroylsarcosine), and U.S. Pat. No. 4,975.529, which uses2-amino-2-methyl-1-propanol. Each of these patents are incorporatedherein by reference. Although SDS has been reported in severalreferences for use in somatotropin solubilization and naturationmethods, it is further acknowledged that SDS binds relatively tightly tothe naturated somatotropin, thus making its complete removal from thesomatotropin solution difficult.

There is a need in the art for more economical and efficient methods toobtain recombinant somatotropins with high yield and purity. Inparticular, there is a need for methods for the solubilization andnaturation of recombinant somatotropin proteins to obtain thesomatotropin molecules in a bioactive state, preferably using a lowamount of detergent that is readily biodegradable. There is a furtherneed for such methods that use a detergent that is easily removed fromthe naturated somatotropin.

SUMMARY OF THE INVENTION

This invention generally relates to methods for producing biologicallyactive recombinant somatotropins. More particularly, this inventionrelates to methods for the solubilization and naturation of recombinantsomatotropins comprising contacting a somatotropin with a detergentcomposition and water under conditions effective to obtain a naturatedsomatotropin, wherein the detergent composition comprises a C₁₀ to C₁₈acyl glutamate, a C₁₀, C₁₄ or C₁₈ alkyl sulfate, an alcohol ethoxysulfate, lauroyl ethylenediaminetriacetic acid (LEDA), a C₁₀ to C₁₈linear alkyl benzene sulfonate, diphenyl disulfonate or an acyl aminoacid of formula (I) or formula (II):

wherein A is CH₂CH₂CO₂H, CH₂CH₂SCH₃, CH₂CO₂H, CH₂CONH₂, CH₂CH₂CONH₂,CH(CH₃)₂, CH₂CH(CH₃)₂, CH(CH₃)CH₂CH₃, CH₂C₆H₅, CH₂C₆H₄OH or CH₂OH and mis an integer from 8 to 16;

 CH₃(CH₂)_(m)CONH(CH₂)_(n)CO₂H  (II)

wherein n is an integer from 1 to 5 and m is an integer from 8 to 16.

In a further preferred embodiment, the invention is directed to a methodfor the solubilization and naturation of a recombinant somatotropinusing low amounts of biodegradable detergents that are easily removedfrom the naturated somatotropin by diafiltration, such as a C₁₀ or C₁₂acyl glutamate, N-lauroyl sarcosine, N-decylsulfate (NDS) or lauroylethylenediamine-triacetic acid (LEDA).

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention is directed to a method for the naturation of asomatotropin comprising the step of contacting a somatotropin with adetergent composition and water under conditions effective to obtain anaturated somatotropin, wherein the detergent composition comprises aC₁₀ to C₁₈ acyl glutamate, a C₁₀, C₁₄ or C₁₈ alkyl sulfate, an alcoholethoxy sulfate, lauroyl ethylenediaminetriacetic acid (LEDA), a C₁₀ toC₁₈ linear alkyl benzene sulfonate, diphenyl disulfonate or an acylamino acid of the formula (I) or (II):

wherein A is CH₂CH₂CO₂H, CH₂CH₂SCH₃, CH₂CO₂H, CH₂CONH₂, CH₂CH₂CONH₂,CH(CH₃)₂, CH₂CH(CH₃)₂, CH(CH₃)CH₂CH₃, CH₂C₆H₅, CH₂C₆H₄OH, or CH₂OH and mis an integer from 8 to 16;

 CH₃(CH₂)_(m)CONH(CH₂)_(n)CO₂H  (II)

wherein n is an integer from 1 to 5 and m is an integer from 8 to 16.The detergent composition can also comprise an N-alkylated derivative offormula (I) or (II).

The invention is further directed to a method for the solubilization andnaturation of a somatotropin comprising the steps of contacting asomatotropin with a detergent composition and water under conditionseffective to solubilize the somatotropin, and subsequently adjusting thepH of the resulting somatotropin solution to naturate the somatotropin,wherein the detergent composition comprises a C₁₀ to C₁₈ acyl glutamate,a C₁₀, C₁₄ or C₁₈ alkyl sulfate, an alcohol ethoxy sulfate, lauroylethylenediaminetriacetic acid (LEDA), a C₁₀ to C₁₈ linear alkyl benzenesulfonate, diphenyl disulfonate or an acyl amino acid of the formula (I)or (II):

wherein A is CH₂CH₂CO₂H, CH₂CH₂SCH₃, CH₂CO₂H, CH₂CONH₂, CH₂CH₂, CONH₂,CH(CH₃)₂, CH₂CH(CH₃)₂, CH(CH₃)CH₂CH₃, CH₂C₆H₅, CH₂C₆H₄OH, or CH₂OH and mis an integer from 8 to 16;

CH₃(CH₂)_(m)CONH(CH₂)_(n)CO₂H   (II)

wherein n is an integer from 1 to 5 and m is an integer from 8 to 16.The detergent composition can also comprise an N-alkylated derivative offormula (I) or (II).

For purposes of the present invention, the following terms should beconsidered to have the definitions listed below.

The term “somatotropin” includes mammalian somatotropins, such asbovine, porcine, human, ovine, equine, canine and feline somatotropin,and others such as avian somatotropin. Somatotropins, for purposes ofthis specification, includes somatotropin proteins havingnaturally-occurring sequences, analogs and homologs of thenaturally-occurring protein having somatotropin-like bioactivity, i.e.,they bind to somatotropin receptors in the animal with affinity greatenough to enhance juvenile growth rate, lactation and/or feedefficiency. Somatotropins also include variants of thenaturally-occurring somatotropin that have been lengthened, shortened,substituted and/or fused to another protein, provided that such variantsare subject to solubilization and/or naturation according to theinventive process.

“Recombinant” proteins, also referred to as heterologous proteins, areproteins which are normally not produced by the host cell. RecombinantDNA technology has permitted the expression of relatively large amountsof heterologous proteins from transformed host cells. For example,expression of recombinant somatotropins from a variety of animals bytransformed microorganisms is known. Examples include Goeddel, et al.,“Direct Expression in Escherichia coli of a DNA Sequence Coding forHuman Growth Hormone,” Nature 281:544-48 (1979) and Seeburg, et al.,“Efficient Bacterial Expression of Bovine and Porcine Growth Hormones,”DNA 2:37-45 (1983). The production of somatotropins in transformedmicroorganisms can be achieved by a variety of recombinant geneticplasmids. Examples include those described in United Kingdom PatentApplication GB 20732445A; and Schoner et al., “Role of mRNATranslational Efficiency in Bovine Growth Hormone Expression inEscherichia coli,” PNAS USA 81:5403-07 (1984). Analogs of bST are alsoknown, for example, as disclosed in European Patent Application No.103,395. The production of bST in transformed microorganisms other thanE. coli has been reported by Gray, et al., “Synthesis of Bovine GrowthHormone by Streptomyces lividans,” Gene 32:21-30 (1984), and in U.S.Pat. No. 4,443,539 for yeast.

“Inclusion bodies,” also referred to as “refractile bodies,” are thecytoplasmic aggregates and oligomers containing the recombinantsomatotropin to be recovered.

A “host cell” is a microbial cell such as bacteria, yeast or othersuitable cells such as animal and plant cells that has been transformedto express the recombinant somatotropin. An exemplary host cell is E.coli K12 (strain W3110/pBGH-1) which has been transformed to permitexpression of bovine somatotropin.

“Naturation” involves the formation of correct disulfide bonds such thatthe somatotropin protein is biologically active upon completion of thenaturation step or after further purification. “Folding” refers to thereturn of the overall conformational shape of the protein sufficient topermit proper oxidation. “Oxidation” refers to the formation of theintramolecular disulfide bonds generally required for a biologicallyactive conformation, preferably the native biologically activeconformation.

“Biological activity” means that the subject somatotropin is capable ofeffecting its intended in vivo physiological response. The biologicalactivity can be determined in the absence of in vivo testing in theparticular species by carrying out suitable bioassays. A suitablebioassay for the somatotropins is the “rat weight gain bioassay.” Inthis bioassay, the bioactivity of somatotropin preparations are assessedrelative to a known preparation (i.e., extracted native somatotropin) byrelating the amount of weight gain of hypophysectomized rats to varyingamounts of administered preparation.

The methods for solubilization and naturation of somatotropins accordingto the invention may be applied to any type of recombinant somatotropin,particularly bovine somatotropin (bST), porcine somatotropin (pST) andhuman somatotropin (hST). The transformation, culturing and fermentingof host cells to produce recombinant somatotropin may be performed byconventional methods. The recombinant somatotropin, generally in theform of inclusion bodies, may also be recovered from the host cellculture by conventional techniques that disrupt the cell so as torelease the inclusion bodies, and thereupon the inclusion bodies may becollected as a pellet by differential centrifugation.

The detergent composition to be used in the inventive method generallycontains a detergent or combination of detergents that promotes thesolubilization of somatotropin obtained from the host cell and/ornaturates the somatotropin molecules. More preferably, the detergentused in the inventive methods is also easily removed from thesomatotropin solution upon completion of the naturation step. Thedetergent composition to be used in the inventive methods may compriseone or more compounds selected from a C₁₀ to C₁₈ acyl glutamate, a C₁₀,C₁₄ or C₁₈ alkyl sulfate, an alcohol ethoxy sulfate, lauroylethylenediaminetriacetic acid (LEDA), a C₁₀ to C₁₈ linear alkyl benzenesulfonate, diphenyl disulfonate or an acyl amino acid of the formula (I)or (II):

wherein A is CH₂CH₂CO₂H, CH₂CH₂SCH₃, CH₂CO₂H, CH₂CONH₂, CH₂CH₂CONH₂,CH(CH₃)₂, CH₂CH(CH₃)₂, CH(CH₃)CH₂CH₃, CH₂C₆H₅, CH₂C₆H₄OH, or CH₂OH and mis an integer from 8 to 16;

CH₃(CH₂)_(m)CONH(CH₂)_(n)CO₂H  (II)

wherein n is an integer from 1 to 5 and m is an integer from 8 to 16.The detergent composition can also comprise an N-alkylated derivative offormula (I) or (II).

In a preferred embodiment, the detergent is a C₁₀ to C₁₈ acyl glutamateor a salt thereof, and more preferably a C₁₀, C₁₂, C₁₆ or C₁₈ acylglutamate or a salt thereof. For example, the acyl radical may becocoyl, lauroyl, stearoyl or a mixture thereof. In a preferredembodiment, the acyl glutamate is an acyl glutamate salt oftriethanolamine sodium or potassium. More preferably, the acyl glutamateis sodium lauroyl glutamate (available as AMISOFT LS-11 or AMISOFTLS-11(F) from Ajinomoto), sodium cocoyl glutamate (available as AMISOFTCS-11 or AMISOFT CS-11(F) from Ajinomoto), TEA cocoyl glutamate(available as AMISOFT CT-12 or AMISOFT CT-12S from Ajinomoto), TEAlauroyl glutamate (available as AMISOFT LT-12 from Ajinomoto), sodiumhydrogenated tallowyl glutamate (available as AMISOFT HS-11 or AMISOFTHS-11(F) from Ajinomoto), sodium hydrogenated tallowyl glutamate withsodium cocoyl glutamate (available as AMISOFT GS-11 or AMISOFT GS-11(F)from Ajinomoto), disodium hydrogenated tallowyl glutamate (available asAMISOFT HS-21 from Ajinomoto), potassium cocoyl glutamate (available asAMISOFT CK-11 or AMISOFT CK-11(F) from Ajinomoto), cocoyl glutamic acid(available as AMISOFT CA from Ajinomoto) or hydrogenated tallowylglutamic acid (stearoyl glutamic acid) (available as AMISOFT HA fromAjinomoto). More preferably, the detergent composition comprises an acylglutamate of sodium lauroyl glutamate, sodium hydrogenated tallowylglutamate, sodium cocoyl glutamate, disodium cocoyl glutamate or acombination thereof. Most preferably, the detergent compositioncomprises a C₁₀ or C₁₂ acyl glutamate, and in particular, sodium lauroylglutamate.

The detergent can also be an N-alkylated derivative of formula (I) or(II), such as acyl N-methyl glutamate, acyl N-methyl aspartate or anN-methyl derivative of Sarkosyl or Amilite.

Acyl glutamate can be added to the subject somatotropin mixture, forexample, as a powder or in solution form. For example, acyl glutamatecan be prepared as a 10% stock solution having a pH of about 6.9-7.0 byadding 0.84 L H₂O, 61 mL 10% NaOH, and 100 g of acyl glutamate powder.During the solubilization and naturation steps, acyl glutamate ispreferably present in the overall mixture at a concentration in therange of about 0.2 to about 5% weight based on the total naturationmixture, and more preferably about 0.3 to about 3%.

The use of acyl glutamates in the detergent composition in the inventivemethods provides a number of economical and environmental advantages, asacyl glutamates are generally available at low cost and high purity andare readily biodegradable. The use of certain acyl glutamates also addsto the overall economics of the inventive methods as they are easilyseparated from the mixture of naturated somatotropin, particularly bydiafiltration, thereby reducing equipment and operation costs.

Alternatively, the detergent composition used in the inventive methodscomprises a C₁₀ C₁₄ or C₁₈ alkyl sulfate, such as NDS, an alcohol ethoxysulfate, such as C₁₂H₂₅O(CH₂CH₂O)SO₃Na or C₁₂H₂₅O(CH₂CH₂O)₃SO₃Na(available from Stephan Co. as Steol™ CS-130, CS-330 or CS-430) orC₁₄H₂₉O(CH₂CH₂O)₃SO₃Na (available from Henkel Corp. as Standapol™ES-40); LEDA (available from Hampshire Corp. as Hampshire LEDA); a C₁₀to C₁₈ linear alkyl benzene sulfonate, such as a linear dodecylbenzenesulfonate, diphenyl disulfonate or an acyl amino acid of the formula (I)or (II):

wherein A is CH₂CH₂CO₂H, CH₂CH₂SCH₃, CH₂CO₂H, CH₂CONH₂, CH₂CH₂CONH₂,CH(CH₃)₂, CH₂CH(CH₃)₂, CH(CH₃) CH₂CH₃, CH₂C₆H₅, CH₂C₆H₄OH, or CH₂OH andm is an integer from 8 to 16;

CH₃(CH₂)_(m)CONH(CH₂)_(n)CO₂H   (II)

wherein n is an integer from 1 to 5 and m is an integer from 8 to 16.

The solubilization and naturation steps may be conducted at the samevolume and detergent concentration. Alternatively, the solubilizationstep may proceed at less than the final naturation volume assolubilization of the subject protein may be facilitated at higherdetergent concentrations. The somatotropin solution may then be dilutedto obtain the final naturation volume. For example, the somatotropin maybe solubilized in a detergent solution having half of the finalnaturation volume, after which an amount of water or buffer is added toobtain the full naturation volume.

The pH of the solubilization step is preferably in the range of about 9to about 13, and more preferably in the range of about 10 to about 12.The pH of the naturation solution is preferably in the range of about 8to about 12, more preferably about 9 to about 11 and most preferablyabout 9.5 to about 10.5.

The naturated somatotropin obtained by the inventive methods ispreferably obtained in at least about 80% yield, for example in a yieldof about 85% to 95%, wherein the yield is defined as [naturatedsomatotropin monomer]/[total reduced somatotropin monomer].

A catalyst, such as cystine or cysteine, may be optionally added to thesomatotropin composition of the naturation step to increase the rate offormation of the disulfide bonds.

The detergent composition used for solubilization and/or naturation ofthe somatotropin is preferably removed from the somatotropin solutionafter the naturation step to avoid interference with downstreampurification steps. The detergent composition may be removed by avariety of methods including ion-exchange, dialysis or combinations ofthose techniques. In a preferred embodiment, the detergent is removedfrom the naturated somatotropin composition by diafiltration. In afurther preferred embodiment, the diafiltration is conducted with acellulose membrane. Removal of detergents such as C₁₀ and C₁₂ acylglutamates, N-lauroyl sarcosine, NDS or LEDA by diafiltration is asignificant processing advantage as certain detergents are removed fromthe somatotropin mixture with a significantly lower number of turnovervolumes than known processes. When some detergents are removed bydiafiltration alone, they may require in excess of 80 volumes ofdiafiltration buffer. This large volume is a disadvantage in processingbecause of the size of the diafiltration equipment required. Incontrast, C₁₀ and C₁₂ acyl glutamates, N-lauroyl sarcosine, NDS, andLEDA have been found to be easily removed with preferably fewer than 30turnover volumes. C₁₀ and C₁₂ acyl glutamates are particularly amenableto removal by diafiltration and are more preferably removed in about 10to 20 turnover volumes.

Precipitation of impurities and purification of the naturatedsomatotropin composition having the detergent composition substantiallyremoved may be carried out by conventional methods. For example, Storrset al., U.S. Pat. No. 5,182,369 and Bentle et al., U.S. Pat. No.4,652,630, which are incorporated herein by reference, refer to methodsfor purifying and recovering biologically active somatotropin monomersfrom solution following the solubilization and naturation of inclusionbodies of host cells produced by recombinant DNA methodology.

The methods of the present invention are preferably part of an overalltechnique for producing a somatotropin product that is suitable forparenteral application to target animals.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

EXAMPLES

Four varieties of acyl glutamate and lauroyl ethylenediaminetriaceticacid (LEDA) were tested and shown to be highly efficient at solubilizingbovine somatotropin (bST) inclusion bodies and allowing the correctnaturation of the bST molecules (Examples 1-3). Examples are alsoprovided for the solubilization and naturation of porcine somatotropin(pST) (Examples 3-4).

Example 1

Solubilization of bST Inclusion Bodies

An aqueous slurry of bST inclusion bodies was assayed and determined tocontain 45 g bST/L. The bST used was the N-methionyl derivative of bSTotherwise having the native amino acid sequence beginning withphenylalanine. These solubilization experiments were conducted at roomtemperature, a solution pH of 11.5 and at 20 g bST/L. The solubilizationwas performed at one half of the naturation volume, i.e., if thenaturation was to be carried out in a 1% detergent and at 10 g bST/L,the solubilization was carried out in a 2% detergent and at 20 g bST/L.The volume was doubled after the solubilization to start the naturation.

Water, detergent and NaOH were combined. A sufficient amount of the basewas added such that after the inclusion bodies were added and dissolved,the pH was 11.5, as determined in a preliminary experiment. The mixtureof water, detergent and base was stirred as the inclusion bodies wereadded, after which stirring was continued for 30 minutes.

Example 2

Naturation of bST

The naturation step was also conducted at room temperature. Followingthe solubilization step, the pH was adjusted to 9.5 by adding HCl, whichalso served to nearly double the volume. Water was then added to bringthe solution to its final volume. Cystine was then added to achieve afinal concentration of 1 mM cystine. The mixture was then stirredovernight to complete the naturation process.

Examples 1 and 2 were conducted for four different acyl glutamatedetergent compositions, sodium lauroyl glutamate, sodium hydrogenatedtallowyl glutamate with sodium cocoyl glutamate, sodium hydrogenatedtallowyl glutamate and disodium cocoyl glutamate, and LEDA as well as acomparative detergent of sodium lauroyl sarcosinate. Tables 1 and 2below summarize the naturation efficiencies a various pH conditions anddetergent concentrations.

TABLE 1 Naturation Efficiencies (%) for Solubilization pH of 11.5 andNaturation pH of 9.5 Detergent Concentrations DETERGENT 0.05 0.10 0.250.50 1.00 1.50 2.00 2.50 Sodium lauroyl — — — — — 90 — — glutamateSodium — — — 93 94 75 37 23 hydrogenated tallowyl glutamate + sodiumcocoyl glutamate Sodium — 54 76 95 88 57 25 15 hydrogenated tallowylglutamate Disodium — — — 63 81 88 69 32 cocyl glutamate Lauroyl — — — —— — 67 83 ethylenedi- aminetriacetic acid Sodium lauroyl 28 37 43 89 6550 45 45 sarcosinate* *Comparative example

TABLE 2 Naturation Efficiencies (%) at Various Naturation pH ValuesRefold pH DETERGENT 8.5 9.5 10.5 11.5 Sodium lauroyl 77 90 85 79glutamate Sodium 84 95 97 82 hydrogenated tallowyl glutamate Disodiumcocoyl 71 88 88 76 glutamate Lauroyl 68 83 76 69 ethylenediaminetriacetic acid Sodium lauroyl 81 89 81 67 sarcosinate* *Comparativeexample

Example 3

Solubilization and Naturation of pST

PST was solubilized at half of the ultimate naturation volume in 3%lauroyl acylglutamate, 20 g/L pST, pH 11 to 11.5 at room temperature.The pST used was that having the native amino acid sequence beginningwith phenylalanine and having an additional alanine at the aminoterminus. Following solubilization, 15 to 30 min., the pH was adjustedwith HCl, diluted with deionized water to final naturation volume,followed by addition of the cystine catalyst. Final naturationconditions were 10 g/L pST. 1.5% acylglutamate, 1 mM cystine, pH 9.5, atroom temperature. The naturation was completed within a few hours.

Example 4

Removal of Acyl Glutamate Detergent From Naturated pST

The lauroyl acyl glutamate detergent used in the naturation step wasremoved by diafiltration prior to pH precipitation to remove impurities.The acylglutamate was adequately removed with 15 to 20 turnover volumesof cold diafiltration buffer of 1 mM NaOH in deionized water.

While the methods of this invention have been described in terms ofpreferred embodiments, it will be apparent to those of skill in the artthat variations may be applied to the method described herein withoutdeparting from the concept, spirit and scope of the invention. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention.

What is claimed is:
 1. A method for the naturation of a somatotropincomprising: contacting a somatotropin with a detergent composition inwater to provide a somatotropin solution at pH of about 8 to about 13,the detergent composition consists essentially of C₁₀ alkyl sulfatepresent at about 0.2% to about 5%, by weight, of the total solution;and, removing the detergent from the naturated somatotropin solution bydiafiltration.
 2. The method of claim 1, the somatotropin is bovine,porcine or human somatotropin.
 3. The method of claim 1, the naturatedsomatotropin yield is about 80% or higher.
 4. The method of claim 1,further adjusting the pH of the solution from about 8 to about
 12. 5.The method of claim 4, further adjusting the pH of the solution fromabout 9.5 to about 10.5.
 6. A method for solubilization and naturationof a somatotropin comprising: contacting a somatotropin with a detergentcomposition in water to provide a somatotropin solution at pH of about 9to about 13, the detergent composition consists essentially of C₁₀ alkylsulfate at about 0 2% to about 5% by weight, of the total solution; andremoving the detergent from the naturated somatotropin solution bydiafiltration.
 7. The method of claim 6, the somatotropin is bovine,porcine or human somatotropin.
 8. The method of claim 6, the naturatedsomatotropin yield is about 80% or higher.
 9. The method of claim 6,further adjusting the pH of the somatotropin solution from about 8 toabout
 12. 10. The method of claim 9, further adjusting the pH of thesomatotropin solution from about 9.5 to about 10.5.
 11. A method forobtaining a bioactive somatotropin comprising: contacting a somatotropinwith a detergent composition in water to provide a somatotropin solutionat pH about 9 to about 13, removing the detergent composition from thenaturated somatotropin solution by diafiltration, the detergentcomposition consists essentially of N-decylsulfate at about 0.2% toabout 5%, by weight, of the total solution.
 12. The method of claim 11,the diafiltration uses fewer than 30 turnover volumes for removing thedetergent from the somatotropin.
 13. The method of claim 12, thediafiltration uses from 10 to 20 turnover volumes for removing thedetergent from the somatotropin.